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Prostaglandins & other Lipid Mediators 113–115 (2014) 2–12
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Prostaglandins and Other Lipid Mediators
eview
he role of long chain fatty acids and their epoxide metabolites inociceptive signaling
aren Wagner, Steve Vito, Bora Inceoglu, Bruce D. Hammock ∗
epartment of Entomology and UC Davis Comprehensive Cancer Center, University of California Davis, Davis, CA 95616, United States
r t i c l e i n f o
rticle history:vailable online 18 September 2014
eywords:mega-3 fatty acidspoxy fatty acids (EpFAs)
a b s t r a c t
Lipid derived mediators contribute to inflammation and the sensing of pain. The contributions of omega-6 derived prostanoids in enhancing inflammation and pain sensation are well known. Less well exploredare the opposing anti-inflammatory and analgesic effects of the omega-6 derived epoxyeicosatrienoicacids. Far less has been described about the epoxidized metabolites derived from omega-3 long chainfatty acids. The epoxide metabolites are turned over rapidly with enzymatic hydrolysis by the soluble
poxyeicosatrienoic acids (EETs)poxydocosapentanoic acids (EDPpDPEs)oluble epoxide hydrolase (sEH)ain
epoxide hydrolase being the major elimination pathway. Despite this, the overall understanding of therole of lipid mediators in the pathology of chronic pain is growing. Here, we review the role of longchain fatty acids and their metabolites in alleviating both acute and chronic pain conditions. We focusspecifically on the epoxidized metabolites of omega-6 and omega-3 long chain fatty acids as well as anovel strategy to modulate their activity in vivo.
Published by Elsevier Inc.
ontents
1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31.1. PUFA metabolism. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31.2. EpFA synthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31.3. EpFA metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31.4. Lipids in nociception . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
2. Parent PUFAs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42.1. Antinociceptive activity of parent PUFAs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42.2. Inflammatory pain. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52.3. Neuropathic pain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52.4. Other biologies of parent PUFAs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
3. EpFA metabolites. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63.1. Antinociceptive activity of EpFA metabolites . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63.2. Inflammatory pain. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63.3. Neuropathic pain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63.4. Other biologies of EpFA metabolites . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
4. Soluble epoxide hydrolase inhibitors (sEHI) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 84.1. Antinociceptive activity of sEHI . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 84.2. Inflammatory pain. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
4.3. Neuropathic pain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4.4. Other biologies of sEHI . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5. Translational pathways . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
∗ Corresponding author at: Department of Entomology, University of California Davis, Oax: +1 530 752 1537.
E-mail address: [email protected] (B.D. Hammock).
ttp://dx.doi.org/10.1016/j.prostaglandins.2014.09.001098-8823/Published by Elsevier Inc.
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ne Shields Avenue, Davis, CA 95616, United States. Tel.: +1 530 752 7519;
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6. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10Funding . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10Conflict of interest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
. Introduction
The role of arachidonic acid (ARA) metabolites in pain andnflammation has received great attention over the last 45 years.his has expanded the understanding of lipids as cell signalingolecules with biological actions not limited to their role as
nergy stores. The exploration of bioactive lipid metabolites hasargely remained focused on the omega-6 polyunsaturated fattycid (PUFA) arachidonic acid ARA. Recently, this has extended tonvestigating the biological fate of the omega-3 fatty acids throughhe same enzymatic pathways of the ARA cascade. The omega-6nd omega-3 PUFAs differ in the position of the double bonds fromhe methyl end of the molecules. Nevertheless, both are formedrom the essential fatty acids linoleic acid and �-linolenic acidespectively which cannot be synthesized de novo by humans orther mammals. However, the conversion of �-linolenic acid toong chain omega-3 PUFAs is limited, and they are more efficientlyaken in through dietary supplementation [1]. The PUFAs affect
embrane fluidity, modulate inflammation, hemostasis and vascu-ar tone [2]. Roles for omega-3 PUFAs in the central nervous systemCNS) and CNS development have been well described [3,4] and
ore recently the role in CNS pathology has attracted focus [5,6].ere we examine the bioactivity of these omega-3 PUFAs compared
o omega-6 ARA and their metabolites with special attention paid toheir role in modulating nociceptive signaling and pain. While newvidence is emerging regarding the role of omega-3 PUFA metabo-ites of lipoxygenase enzymes in pain [7], this review will focus onhe epoxidized PUFA metabolites (epoxy fatty acids, EpFAs) formedy cytochrome P450 enzymes.
.1. PUFA metabolism
The ARA cascade is typically simplified and described as threenzymatic pathways including the cyclooxygenase (COX), lipoxy-enase (LOX) and cytochrome P450 (CYP450) enzyme families thatonvert the parent PUFAs to multiple bioactive lipid metabolites.t is now established that the omega-3 fatty acids, both eicosapen-aenoic acid (EPA, 20:5 n-3) and docosahexaenoic acid (DHA, 22:6-3), in addition to omega-6 ARA (20:4 n-6), are substrates for eachf the major catalyzing enzymes of these three branches of the cas-ade [8,9]. While use of the parent PUFAs as dietary interventionas been more often investigated, there is a broadening explorationf the activity of individual metabolite species. The COX producedrostaglandins from ARA are well studied and prostaglandin E2pecifically is known to have a prominent role in pain and inflam-ation [10]. The COX-2 enzyme acts on EPA and DHA to form the
espective E and D series resolvins in the presence of aspirin, and theOX enzymes form neuroprotectin-1 from DHA [11,12]. The LOXroduced leukotrienes from ARA have roles in inflammation and
ung related pathologies such as asthma [13]. The CYP450 producedetabolites from ARA are less well described and their biological
ctivity is still being explored. The CYP450 oxidases also act on themega-3 substrates forming multiple species of EpFAs [14] as wells numerous hydroxylated metabolites. Thus, a new nomenclatureuch as the PUFA cascade is more applicable.
present in neurons and glial cells in both rodents and humans[15,16]. The omega-6 ARA and omega-3 EPA and DHA PUFAs arecellular membrane lipids esterified primarily at the sn-2 position ofglycerophospholipids [9,17,18]. All three classes are released fromthese stores by phospholipases [19] and other enzymes. They aresubsequently transformed by CYP450 enzymes into regioisomersof the EpFAs, the epoxydocosapentanoic acids (EDPs also knownas EpDPEs), eicosatetraenoic acids (EEQs also known as EpETEs)and epoxyeicosatrienoic acids (EETs) from DHA, EPA and ARArespectively. The CYP450 oxidases convert all of these substrateswith higher catalytic turnover of EPA and DHA to ARA [20,21].CYP2C8, CYP2C9 and CYP2J2 are reported to be largely responsi-ble for the formation of the EpFAs. However several other P450oxidases thought to be principally responsible for the oxidativemetabolism of the lipophilic xenobiotics may also produce EpFAs.All of these CYP450s make several regioisomers of EpFAs and all ofthem are involved to varying degrees in allylic, omega, and omega-1 hydroxylation in addition to epoxidation. The PUFA substratescan be epoxidized at any of several double bonds resulting in sev-eral regioisomer metabolites and the corresponding enantiomersfor each class of PUFAs. Thus, there is a large metabolite pool whenall possible outcomes are considered. EETs and omega-3 EpFAsare also esterified in phospholipids present in cellular membranesand plasma lipoproteins [22–24]. Therefore, they are immediatelyavailable upon release from these membranes.
1.3. EpFA metabolism
EETs are endogenous substrates of the soluble epoxide hydro-lase (sEH, EPHX2, EC 3.3.2.10) an enzyme downstream of theCYP450s in the ARA cascade. The sEH appears primarily respon-sible for the hydration of EpFAs to their corresponding diols whenit is present. These endogenous substrates are efficiently degradedby the sEH making it an important regulatory enzyme (Fig. 1). Inhis classical review of the epoxide hydrolases Oesch pointed outthat the microsomal epoxide hydrolase (mEH, EPHX1, EC 3.3.2.9)can hydrolyze epoxysteric acid [25]. The hydrolysis of fatty acidepoxides by the mEH has been observed by numerous subsequentworkers [26,27], however compared to sEH the Km is higher, theVmax or kcat values are much lower, and in general the abundanceof the mEH is much lower. Thus, except possibly in rare tissueswhere the sEH level is exceptionally low and mEH level is high,the sEH is the predominant enzyme responsible for the hydrolyticdegradation of fatty acid epoxides. However, other mechanismssuch as reincorporation into the cellular membrane and beta oxi-dation also limit the in vivo residence time of EETs. Several studieshave attempted to determine the maximum possible hydrolysis ofEpFAs in tissues that is not due to the sEH ranging from the earlywork of Moody to more recent investigations [28,29]. The conclu-sion is that no substantial hydrolysis of EpFAs is due to enzymesother than the sEH. There are several genes that could code for pro-teins with sEH activity, with EH3 and EH4 the most well studied[30]. While there are reports of EpFA hydrolysis by EH3 [31] manylaboratories have difficulty in reproducing this work possibly dueto expression problems.
The sEH converts EpFAs of all three classes into the vicinal
.2. EpFA synthesisThe enzymes responsible for the de novo production and degra-ation of EpFAs are broadly distributed through tissues and are
diols. There is evidence of substrate preference, for example sEHhas the strongest preference for the 14,15 isomer of the EETs, andincreasing the distance between the terminal carbon and the epox-ide reduces this preference [23]. However, omega-3,4 epoxides of
4 K. Wagner et al. / Prostaglandins & other Lipid Mediators 113–115 (2014) 2–12
Fig. 1. Omega-6 and omega-3 long chain PUFAs share a similar metabolic fate. The fatty acids are designated based on the position (3 or 6) of the first double bondfrom the methyl (omega, �) end of the molecule. Both omega-6 arachidonic acid (ARA) and omega-3 eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) aremetabolized by cytochrome P450 oxidases. Several epoxy fatty acid regioisomer metabolites the epoxyeicosatrienoic acids (EETs), epoxyeicosatetraenoic acids (EEQs) andepoxydocosapentaenoic acids (EDPs) respectively, and their enantiomers are possible for each of the substrates based on which double bond is epoxidized. Arachidonica oxidizw ids (Da cludin
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cid, for example, may form the 5–6, 7–8, 11–12, or 14–15 EET (pictured). The ephich converts them to their vicinal diols termed the dihydroxyeicosatrienoic ac
cids (DiDPAs) respectively. The EpFAs have multiple beneficial biological effects in
he EDPs and EEQ series are slowly metabolized [32]. Despite thiselectivity, sEH turns over all of the regioisomers. The EETs haveeen shown to regulate vascular tone and blood pressure as wells being anti-inflammatory and antihyperalgesic in rodent mod-ls of disease [33–36]. The hydrated diol products produced fromRA by sEH, the dihydroxyeicosatrienoic acids (DHETs), are thought
o have little or no biological activity [20,37]. The diols are moreolar, they exit the cells more quickly and are rapidly conjugated38]. However, there is evidence that the DHETs facilitate chemoat-raction of monocytes [39]. Diols of linoleic acid (leukotoxin andsoleukotoxin diols) increase vascular permeability, cause edema,nd appear to be powerful proinflammatory chemical mediators40]. Clear biological activity for the diols of the omega-3 EpFAs hasot been documented [32], and they are expected to demonstrate aimilar increase in solubility and elimination to the omega-6 dihy-roxy products.
.4. Lipids in nociception
Here, we examine the role of these lipids in antinociceptionxamining evidence in both acute and chronic pain conditions.ociception is the action of the nervous system in encoding noxious
timuli that results in the sensation of pain and antinociception islocking this action. The differences between acute tissue injuryr inflammation and unresolved neuropathic pain are examinedn several reviews [41–43]. It is known clinically that some phar-
acological therapies for pain such as NSAIDs and opiates thatre active against inflammatory hyperalgesia (an increased painensation from normally painful stimuli) have no or limited effi-acy in chronic pain [44]. This difference in analgesic efficacy isot dependent on peripheral pharmacology because many agentshat act centrally against nerve injury such as gabapentinoids and
nticonvulsants are also effective against tissue injury. The strat-gy of treating painful conditions by modulating lipids has greatherapeutic potential because they are active against both acutend chronic pain. Additionally, altering substrate pools of bioactiveed fatty acids are subject to further metabolism by the soluble epoxide hydrolaseHETs), dihydroxyeicosatetraenoic acids (DHEQs) and dihydroxydocosapentaenoicg mediating analgesia while the diols are thought largely to lack these effects.
molecules to obtain pain relief seems to have few adverse sideeffects [45,46] and therefore the ability to chronically dose painconditions. So far the loss of efficacy (tachyphylaxis) often seenwith some modulators of receptors has not been observed with themodulation of lipid chemical mediators.
2. Parent PUFAs
Recently there is increasing interest in the historical change inlipid consumption revealing a heavily biased omega-6 to omega-3 Western diet [5,47]. The bias was increased in favor of omega-6lipids when omega-3 lipids were removed from industrial cookingoils to increase their thermal stability to oxidation. This dietary biasis important because dietary omega-6 and omega-3 PUFAs competewith each other both for incorporation into cellular membranesand as substrates for enzymes [48]. It has also been demonstratedthat DHA and EPA omega-3 PUFAs cross into the brain efficientlyby simple diffusion [49]. Therefore, omega-3 enriched diets havebeen examined for their effects on pathologies. The results demon-strate that dietary supplementation with the omega-3 PUFAs hasseveral beneficial physiological effects including cardioprotection,reduced inflammation and neuroprotection [50–52]. In addition tothese, omega-3 fatty acid supplementation has been shown to havean effect in various painful conditions including rheumatoid arthri-tis, inflammatory bowel disease, and dysmenorrhea though manyof these effects were interpreted as mostly anti-inflammatory andthereby antihyperalgesic [2]. More recently, the omega-3 fatty acidshave been investigated for their direct antinociceptive effects andpossible mechanisms of the analgesic action, including the gener-ation of active metabolites.
2.1. Antinociceptive activity of parent PUFAs
Earlier studies investigated dietary supplementation of omega-3 rich oils in humans to determine the anti-inflammatory effectof nutritional manipulation on conditions such as rheumatoid
ther Lipid Mediators 113–115 (2014) 2–12 5
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Fig. 2. DHA dietary supplementation improves pain thresholds in neuropathic rats.In a type I diabetic neuropathy model, rats fed an enriched DHA diet (1% DHA cus-tom made diet with Nu-Chek Prep Inc. 99% purity oil) one week prior to diabetesinduction with streptozocin [116] and maintained on the diet for several weeks(pictured day 9–25) showed improved pain thresholds (von Frey assay) comparedto rats on a omega-6 control diet (1% Oleic oil custom made diet with Nu-Chek PrepInc. 99% purity oil) (Two Way Analysis of Variance, Holm Sidak post hoc, ‡p < 0.001groups, *p ≤ 0.030 time points). When both groups were switched to normal chow
K. Wagner et al. / Prostaglandins & o
rthritis [53,54]. Joint pain and stiffness used as endpoints gavenitial evidence that omega-3 intervention could be antihyperal-esic. More recently, the acute effect of omega-3 oil in experimentalodels has been evaluated. Oral dosing has demonstrated reducedrithing in normal mice in the acetic acid assay [55], including doseependent effects after acute enteral administration [56]. Thesebservations have been supported by central intracerebroventric-lar (i.c.v.) administration in mice which significantly reducedrithing behavior [55]. Administration of omega-3 fatty esters also
locked both early and late stages of pain behavior in the forma-in assay in mice [56]. These studies using acute administration ofmega-3 oils in naive mice offer compelling evidence that omega-3atty acids and specifically DHA have antihyperalgesic activity.
Proposed mechanisms of action of omega-3 PUFAs in biologieselated to nociception include the change in cellular membraneipids which alters fluidity and the production of cytokines androstanoids [51]. There is evidence that the omega-3 lipids alterhe activity of ion channels, alter voltage activated sodium cur-ent and displace high affinity ligands at TRPV1 receptors [57–59].dditional results suggests that opioid signaling may be affected,lthough indirectly, with a proposed effect on the release ofndogenous peptides [60]. In humans, dietary supplementationith omega-3 oil increased the downstream levels of EpFA metabo-
ites in plasma [24]. Because the enzymatic metabolism of omega-3atty acids is rapid, it is likely that the bioactive metabolites appearuickly and may be driving the bioactivity of PUFA supplemen-ation.
.2. Inflammatory pain
Inflammation is a complex immune response to high intensitytimulation that results in a release of biological active factors suchs cytokines and peptides from local and migratory cells [61]. Theseactors are able to sensitize and also directly affect sensory neuronsn a way which is recognized as pain [10]. Early work establishedhe parent PUFA ARA has direct pain producing (algogenic) prop-rties [62] and intradermal injection of ARA caused pain in naïveats [63]. A more recent study found that intraplantar administra-ion of ARA in inflamed rats did not further increase pain comparedo the vehicle control [32]. The difference between injecting naïveats and the inflamed rats may be significant, and it is likely thathe effects observed in the naïve rats are overshadowed by thearrageenan induced inflammatory pain state. Intraplantar admin-stration of DHA and EPA also fail to show significant change inhe inflamed model [32]. However, oral omega-3 pretreatment in
odeled inflammatory pain induced by carrageenan demonstratedoth decreased thermal pain and edema [56]. There is also evidencef direct intraarticular administration of DHA in a murine model ofnee arthritis reducing flinching behavior and knee edema [64].ogether the results of these inflammatory pain models supporthe efficacy of omega-3 oil in relieving inflammatory pain.
.3. Neuropathic pain
Neuropathic pain differs from injury or inflammatory painecause it is a chronic pain that lasts beyond injury resolution.ne of the hallmarks of neuropathic pain is that it occurs in thebsence of stimuli [65]. Diabetic neuropathy specifically is a chronicain condition which is a major concern as the worldwide dia-etic population increases, and it is the most common secondaryondition of diabetes mellitus [66]. The type 1 diabetic model ofeuropathy uses the chemical ablation of pancreatic beta islet cells
endering the animals with diabetes and neuropathy resulting fromnchecked hyperglycemia. In diabetic neuropathy, the activity ofesaturase enzymes that produce long chain PUFAs are reducedesulting in diminished levels of DHA and ARA in cellular membranethe improved scores were maintained for up to 10 days (pictured day 25–35) butthen these effects dissipated. Thus, dietary supplementation with the omega-3 PUFADHA improved nociceptive outcomes in a chronic pain model.
phospholipids [67]. Na,K-ATPase activity, a transmembrane sodiumpotassium exchanger enzyme active in the generation of actionpotentials, is decreased in sciatic nerves in modeled diabetic neu-ropathy [68,69]. Omega-3 supplementation partially corrects theactivity of Na,K-ATPase and nerve conduction velocity (NCV) aswell as prevents endoneurial edema and axonal degeneration inthis chronic condition [70]. When tested alone DHA significantlyreduces the decrease in diabetic sciatic NCV and corrects nerveblood flow (NBF) in diabetic rats [71]. However DHA alone didnot have an effect in sciatic nerve Na,K-ATPase activity in theserats. Thus, there is direct effect of DHA distinct from changes inmembrane lipid composition or modulation of Na,K-ATPases thatimproves NCV and NBF.
Recently, omega-3 dietary supplementation has been investi-gated in the diabetic model for effects specifically on nociceptivebehavioral outcomes (Fig. 2). A feeding study using custom dietsof 1% DHA (omega-3) with a corresponding control of 1% oleic acid(omega-6) was conducted in the diabetic neuropathy model. Ratswere fed the respective diets for 1 week prior to the induction ofdiabetes. Following diabetes induction, the withdrawal thresholdsof the animals on the DHA diet were on average higher than controlswhile they were kept on the custom diets. The diets were admin-istered for a total of 25 days before both groups were returned toa conventional chow diet. The pain thresholds of the DHA fed ratsremained higher for an additional week after the switch to conven-tional diet Interestingly, a lasting effect of an increased omega-3to omega-6 epoxyeicosanoid index post cessation of diet supple-mentation was previously observed in humans [21]. Thus, it ispossible the omega-3 metabolite levels remain elevated in rat aswell and would correlate to the nociceptive assay results, thoughthis remains speculation. After this one week period the withdrawalthresholds of the two experimental groups normalized on the reg-ular chow diet demonstrating the improved scores were related to
the DHA supplementation. Thus, a high DHA background is able toimprove nociception as well as improve on microvascular functionsuch as nerve blood flow. The ability to dose omega-3 PUFAs longterm would be a benefit in chronic pain disorders where prolonged
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reatment with many analgesic therapies such as steroids, opioids,nd non-steroidal anti-inflammatory drugs cause severe adverseide effects.
.4. Other biologies of parent PUFAs
The role of omega-3 PUFAs in the central nervous system, espe-ially DHA, is well known in development of the pre and postatal brain. Astrocytes and also hippocampal neurons have demon-trated synthesis of DHA in primary culture from prenatal rat [72].here is growing evidence that this supportive function extends todult brain as well [73]. Experimental and clinical results indicatehat omega-3 supplementation seems beneficial in Alzheimer’s74] as well as major psychiatric disorders such as schizophre-ia [46,75] and select forms of depression [76,77]. Omega-3 PUFAslso have demonstrated effects in neuroprotection and the delay-ng of seizures [78]. Evidence in spinal cord nerve injury suggestsoth acute bolus and dietary omega-3 supplementation are ableo improve survival of motor neurons as well as oligodendrocytesfter compression injury [73]. The pleiotropic effects of omega-
PUFAs in the nervous system underscore their potential formproving CNS structure and function in addition to the specificntinociceptive activity.
. EpFA metabolites
The fate of long chain fatty acids includes oxidation of the PUFAsnto epoxidized bioactive metabolites. Each of the PUFAs gives riseo multiple possible epoxidized regioisomers at the double bondsut also optical isomers (R or S) of each regioisomer resulting in alethora of potentially bioactive metabolites. One significant dif-erence of omega-3 and omega-6 metabolites is that there arelso major proinflammatory metabolites formed from the omega-
ARA. However, the CYP450 generated EETs derived from ARAave consistently demonstrated anti-inflammatory activity. This is
n contrast to ARA metabolites generated by other enzymes suchs the prostaglandins (PGE2, PGD2) and leukotrienes (LTB4) thatre predominately proinflammatory. Thus, although the EETs aremega-6 products, so far similar biology is observed when omega-6ersus omega-3 derived EpFAs are examined. In general the omega-
derived EpFAs appear to be more potent. One notable exceptions that the EETs from ARA are moderately proangiogenic in theresence of VEG-F while the EDPs derived from DHA are stronglyntiangiogenic thus slowing tumor growth and metastasis [79].
The EpFAs, specifically EETs, have well described effects on car-iovascular function but have only recently been investigated forheir role in nociceptive signaling. Most of the COX and LOX pro-uced metabolites of ARA have been found to act on G proteinoupled receptors, many of which have several isozymes [80,81].owever, despite decades of research describing the biologicalffects of EpFAs and in particular EETs, there is currently no knowneceptor for these CYP450 derived metabolites [37]. There haveeen candidates suggested for the receptor and given the diversend tissue dependent biological outcomes it is expected to be oner several GPCRs with multiple isozymes [23]. Despite the lack ofn identified receptor, multiple EETs from ARA and increasinglyDPs from DHA and other EpFA research are demonstrating theirnvolvement in the modulation of multiple biological processes.otably EETs have been shown to be antihyperalgesic in severalain models.
.1. Antinociceptive activity of EpFA metabolites
Potential mechanisms of action of the EET regioisomers in theontext of pain have been investigated using direct microinjec-ion into the ventrolateral periaqueductal gray (vlPAG) [82]. Of
pid Mediators 113–115 (2014) 2–12
the individual EET regioisomers 14,15 EET demonstrated efficacyperipherally as well as the ability to act centrally. Similar to the par-ent PUFA, the 14,15 EET metabolite does not bind opioid receptorsdirectly but alters levels of endogenous opioid peptides. The con-ditional knock out of the CYP450 reductase in neurons halts opioidanalgesia and indicates multiple CYP450 metabolites are possiblyresponsible for this activity [83]. Thus, while there is evidence thatEETs activate TRP channels [84–86], and their application to naïveanimals transiently sensitizes neurons [87], the analgesic activityof EpFAs, specifically EETs, in pain states has been demonstratedrepeatedly.
3.2. Inflammatory pain
The origins of investigating the EpFAs for antinociceptive effectsstemmed from early lipomic analyses revealing one effect of elevat-ing the EETs was a reduction in plasma concentrations of PGE2 [35].Schmelzer et al. followed this observation and found a correlationbetween lowered plasma PGE2 levels and inflammatory pain [88].A regioisomeric mixture of exogenous EETs also blocked inflam-matory pain in the rat model [87]. Later binding studies provideda potential mechanism of action for this analgesia by determin-ing that EETs bind the peripheral benzodiazepine receptor alsoknown as the translocator protein (TSPO) [89]. All four specificregioisomers of EETs were evaluated revealing 14,15 EET and 5,6EET isomers are active. However, TSPO binding has not been deter-mined for EPA or DHA epoxide containing metabolites. The TSPOreceptor contributes to the translocation of cholesterol from theouter to inner mitochondrial membrane by cooperating with thesteroidogenic acute regulatory protein (StarD1). The StarD1 is a car-rier protein that is the rate limiting step in cholesterol delivery tomitochondria for subsequent steroidogenesis. EETs have previouslybeen shown to upregulate StarD1 expression [90]. Thus, EpFAs arehypothesized to modulate neurosteroid synthesis. As such theirantinociceptive effects are reversed by inhibitors of steroid syn-thesis but not by antagonists of 5 major steroid receptors.
The role of EpFAs in mediating antihyperalgesia has also beensupported by topical [91] and intraplantar administration of indi-vidual regioisomers of both EETs and omega-3 derived EpFAs. Bothclasses of chemical mediators significantly improve withdrawalthresholds in models of inflammatory pain [32]. The EpFAs of ARA,EPA and DHA are more effective than their respective parent PUFAs.Additionally, EDPs are the more potent antihyperalgesic mediatorsof the EpFA metabolites. As mentioned earlier omega-3,4 epoxidesare turned over more slowly than other epoxide metabolites of EPAand DHA and seem to be more stable in vivo. For example, the slowermetabolism of the 19,20 EDP may contribute to the relative efficacyof EDP regioisomeric mixtures over other EpFAs. Furthermore, theeffectiveness of different regioisomers within the EDPs has beendetermined. The omega-3 diols tested in the inflammatory painmodel were inactive against nociception further supporting theassignment of the EpFAs as the major biologically active molecules[32].
3.3. Neuropathic pain
To test the effect of the EpFAs in chronic pain, well character-ized regioisomer mixtures of EETs and EDPs were administered in amurine model of type I diabetic neuropathy. The diabetic mice wereassessed for allodynia, a painful response to a normally innocuousstimulus (Fig. 3). While both EpFAs block pain, the EDPs are moreeffective than the EETs in this model. This observation is consistent
with previous evidence demonstrating EDPs have better efficacy invivo against inflammatory pain in the rat [32]. The antihyperalge-sia is rapid but not long lasting because these EpFAs are substratesof the sEH and therefore are subject to rapid degradation in vivo.
K. Wagner et al. / Prostaglandins & other Lipid Mediators 113–115 (2014) 2–12 7
Table 1Summary of outcomes of PUFA and EpFA administration in experimental models.
Pain model Fatty acid Route Species Major outcomes Reference
PUFANaïve (none) n-6 (ARA) int.pl. Rat Intraplantar ARA
painful inRandall-Selitto assay
Gonzales et al. [63]
Inflammatory pain n-3(DHA) Oral Mouse Analgesic againstacetic acid writhing,formalin, tail flick
Nakamoto et al. [55]
n-3 (EPA + DHA) Oral Mouse1, Rat2 Analgesic against aceticacid writhing1andformalin1, effectiveagainst CAR inducedthermal withdrawal2
paw edema2 andperitonitis2
Nobre et al. [56]
n-3(DHA) Oral/int.ar. Mouse Analgesic against CFAarthritis
Torres-Guzman et al.[64]
n-6 (ARA)n-3 (EPA, DHA)
int.pl. Rat No significant changeagainst mechanicalallodynia
Morisseau et al. [32]
Neuropathic pain n-3 (DHA) Diet Rat Analgesic againstmechanical allodynia
Fig. 2
EpFANaïve (none) n-6 (EET) Microinj. vlPAG Rat Analgesic against
thermal tail flickTerashvili et al. [82]
Inflammatory pain n-6 (EET) Dermal Rat Analgesic againstthermal withdrawal inLPS model
Inceoglu et al. [87]
n-6 (EET)n-3 (EDP3,EEQ)
int.pl./i.t.3 Rat Analgesic againstmechanical allodyniain CAR model
Morisseau et al. [32]
Neuropathic pain n-6 (EET)n-3 (EDP)
i.p. Mouse Analgesic againstmechanical allodynia
Fig. 3
int.pl., intraplantar; int.ar., intraarticular; i.p., intraperitoneal; microinj. vlPAG, microinjection of ventrolateral periaqueductal gray; i.t., intrathecal; n-3, omega-3 fatty acids;n-6, omega 6 fatty acids; LPS, lipopolysaccharide, CAR, carrageenan.
1 Corresponds to the LPS model noted to the right in the “Major outcomes” where a sup2 Corresponds to the CAR model noted to the right in the “Major outcomes” where a su
0 15 30 600
20
40
60
80
100
EpDPEsEETs
1mg/kg i.p .
Perc
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f Naï
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Time (minutes )
‡
* #* #
* #
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Fig. 3. EpFAs block chronic neuropathic pain in type I diabetic mice. To test thehypothesis that EpFA metabolites exert analgesic effects the metabolites wereadministered to diabetic neuropathic mice. Type I diabetic neuropathy was inducedwith streptozocin [117] and mice were assessed for neuropathic pain (allodynia)before treatment using the von Frey assay. EDP and EET regioisomeric mixtureswere both dosed at 1 mg/kg administered via intraperitoneal injection (i.p.) and painthresholds were assessed over the time course. Both EpFA mixtures significantlyincreased pain thresholds indicating pain relief (One Way Analysis of Variance,Holm Sidak post hoc, *p ≤ 0.007 EDPs, # p ≤ 0.003 EETs). The EDP regioisomer mix-ture was more effective than the EETs (Mann–Whitney Rank Sum Test, t = 256.0, ‡
p ≤ 0.048) but the effects of both treatments were short lived due to their suspectedrapid degradation by the soluble epoxide hydrolase enzyme. However, exogenousadministration of the EpFA metabolites demonstrates their efficacy in chronic painconditions.
erscript appears.perscript appears.
The acute doses of EpFAs in the chronic pain model define the anti-hyperalgesic effects as distinct from the benefits of longer termomega-3 dietary supplementation on other comorbidities of dia-betes such as hypertension. Moreover, this evidence supports thehypothesis that the EpFAs are directly mediating antihyperalge-sia and are responsible for the antinociceptive activity of PUFAs.Table 1 summarizes the major outcomes of the nociceptive exper-iments outlined in these sections including both the parent PUFAsand the metabolites EpFAs.
3.4. Other biologies of EpFA metabolites
Much of the information on the biochemistry of EpFAs in thebrain relates to EETs. One source of EETs in the CNS are astro-cytes and the excitatory neurotransmitter glutamate can causerelease of EETs from these cells [92,93]. Astrocytes also havedemonstrated epoxide hydrolase activity indicating the tight reg-ulation of these metabolites [94]. EETs are thought to be involvedin K+ signaling by opening large-conductance, calcium activatedK+ channels (BK) in astrocytes [95]. Relevant to nociception andaction in the central nervous system, EETs modulate COX-2 mRNAexpression, regulate cerebral blood flow, and control neurohor-mone release [16,36,96]. Electrical or chemical stimulated cerebralblood flow and tactile sensory stimulated sensory cortex blood floware all blocked by the EET antagonist 14,15-EEZE in vivo suggest-
ing that the effects are mediated by the epoxidized metabolites[97,98]. EETs also increase axonal outgrowth in dorsal root ganglia(DRG) neurons in a concentration dependent manner [99]. Impor-tantly, these results demonstrate that EETs have a direct action in
8 ther Lipid Mediators 113–115 (2014) 2–12
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Fig. 4. The sEHI TPPU improves pain thresholds with enhanced efficacy in omega-3 supplemented neuropathic rats. Similar to the effects of parent PUFA and EpFAadministration, sEHI mediate analgesia. A low 0.3 mg/kg dose of the sEH inhibitorTPPU (structure pictured) demonstrated increased efficacy when administered torats fed a omega-3 DHA enriched diet compared to an omega-6 oleic control dietin the diabetic neuropathy model (Two Way Analysis of Variance, Holm Sidak posthoc, ‡ p ≤ 0.001 groups, *p ≤ 0.050 time points). The DHA fed rats had higher baselinethreshold before subcutaneous (s.c.) TPPU administration, nevertheless their thresh-olds improved over 24% of rats fed an omega-6 control diet (insert). Thus, the use of
K. Wagner et al. / Prostaglandins & o
on-vascular associated cells in the central nervous system. TheETs have demonstrated activity on centrally mediated pain andeizure via intrathecal and i.c.v. injection [36,100]. Picrotoxin, anntagonist of GABA signaling, blocks antinociception mediated bylevated EpFAs and EpFAs demonstrate efficacy altering GABAignaling in chemically but not electrically induced seizure models100,101]. Interestingly, EpFA effectiveness differs in these seizure
odels. Notably, the ARA derived EETs are active against seizureut not the EPA or DHA derived EpFAs. This result is unexpectedecause the EDPs have demonstrated better potency and efficacy
n previous in vitro biochemical endpoints and in vivo nociceptivessays. Despite this, the EpFAs directly affect neuronal and vascularell populations and mediate beneficial outcomes in multiple typesf CNS pathology.
. Soluble epoxide hydrolase inhibitors (sEHI)
The exogenous administration of EpFAs has demonstratedhe direct activity of these metabolites. Another approach is tonhibit the degradation of endogenous EpFAs with small moleculenhibitors of the sEH enzyme. The inhibitors bind the sEH enzymes transition state mimics and this mechanism of action effectivelynhibits the enzyme thereby elevating levels of endogenous EpFAs.nhibiting the sEH has been used as an approach for renal drivenypertension and many other diseases [102]. More recently sEHIave been used to test the role of EpFAs in pain modulation and
or eliciting analgesia mediated by EpFAs [34,103,104]. EpFA lev-ls can be therapeutically increased by sEHI. However, the EpFAsre metabolized by other routes including but not limited to beta-xidation and are reincorporated into cellular membranes. Thisay contribute to the therapeutic index of sEHI which do not have
ubstantial behavioral effects in naïve animals even at high doses102,105].
.1. Antinociceptive activity of sEHI
Many of the results from experiments investigating thentinociceptive effects of EpFAs using exogenous applications ofhe metabolites have been recapitulated with sEHI administration.his strategy has been shown repeatedly to correlate with elevatedpFA levels in plasma and tissues including brain and spinal cords well as in vivo antinociceptive efficacy [32,35,36,100,105]. Andvantage to using sEHI over direct exogenous application of EpFAsr their mimics is that the inhibitors elevate several of the naturallyroduced EpFAs concurrently. In addition, the EpFAs are rapidlyegraded by the sEH, so blocking this enzymatic activity is still
mperative to yielding the maximal beneficial effects of exogenousr endogenous EpFAs. Of course a limitation in the therapeutic usef sEHI is that they only stabilize the EpFAs but do not influenceheir de novo synthesis. This limitation could be overcome by these of stable mimics of EpFA. The mimics, however, may lack theassive therapeutic index associated with sEHI.
.2. Inflammatory pain
The first demonstration of sEHI administration in a pain modelevealed potent antihyperalgesic activity against lipopolysaccha-ide induced inflammatory pain [87]. These experiments demon-trated sEHI mediated decreases in COX-2 protein concomitantith inflammatory prostaglandin decreases [35]. Coadministration
f sEHI with COX-2 selective inhibitors also leads to synergisticncreases in EETs and pain thresholds while decreasing COX-2 pro-
ein and prostaglandins E2 and D2 levels [88]. In the inflammatoryodel sEHI demonstrate down regulation of COX-2 induction in thepinal cords of rats [36]. The potential for synergistic activity haseen further exploited by synthesis of a designed multiple ligand
sEH inhibition with diet supplementation is potential strategy for increasing efficacywhile dose limiting inhibitors for long term dosing in chronic pain conditions.
inhibitor of sEH and COX-2 enzymes resulting in better efficacy innociceptive assays [106]. Related to this, but an important distinc-tion, sEH inhibition attenuates PGE2 induced pain [101]. Blockingpain induced by a direct acting algogen which is also a metabolite ofthe COX enzyme demonstrates that the pain relief is independentof anti-inflammatory properties and also of COX regulation.
The inhibitors have a demonstrated effect on changing the lev-els of spinal somatostatin expression in induced inflammatorypain supporting central effects on nociception [91]. Picrotoxin, aGABA antagonist, blocks sEHI mediated antinociception and theinhibitors are active against seizure induced by this antagonistsuggesting the modulation of GABAergic signaling [100,101]. ThesEHI do not elicit nociceptive change in the absence of a pain state[36,105]. Nevertheless, sEH inhibition modulates COX-2 expres-sion, but it can also block PGE2 induced pain and possibly affectneurosteroid production [101]. It is clear the activity is not limitedto anti-inflammatory mechanisms as demonstrated by the severalexperiments outlined above. However, the positive correlation inall of these indications underscores the idea that the sEHI engagetheir target and are mediating analgesic activity.
4.3. Neuropathic pain
More recently the use of sEH inhibition has extended to neu-ropathic pain models. Similar to the activity of exogenous EpFAsin chronic pain, the sEHI dose dependently relieves allodynia ina model of diabetic neuropathy [36]. The acute administration ofsEHI and the elevation of the EpFAs has no effect on the glycemicstatus of diabetic rats per glucose tolerance, insulin tolerance and
glucose stimulated insulin secretion tests [107]. Thus, inhibitingsEH improves nociceptive outcomes in the diabetic model (alsodemonstrated in Fig. 4), but not by altering metabolomic param-eters. Correlated with improved pain scores, plasma EpFAs levels
ther Lipid Mediators 113–115 (2014) 2–12 9
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Fig. 5. sEHI block the chronic pain of equine laminitis. Laminitis is severe inflamma-tion of the hoof leading to intense pain, tissue destruction, and morbid hypertensionin horses. The condition can be crippling and often fatal. A horse presenting withlaminitis in both front hooves was treated with NSAIDs but suffered refractory pain(Start of Treatment). Therefore the sEHI t-TUCB 0.1 mg/kg i.v. was administered oncedaily for 9 days in addition to the standard of care. After this treatment the horse
K. Wagner et al. / Prostaglandins & o
rom ARA and DHA but not EPA are significantly increased withEH inhibition. However, only EDP to diol metabolite ratios arencreased in spinal cord of animals in this model.
The use of sEHI has been explored at high doses to deter-ine the limits of this approach to analgesia in both inflammatory
nd chronic pain models [105]. The sEHI potently block pain andre not motor impairing even at high doses. However, using lowose sEHI combined with omega-3 dietary supplementation isnother approach for optimizing pain relief with the inhibitors. Thispproach was tested in the type I diabetic neuropathy model. In thisodel DHA diet supplemented neuropathic rats had higher pain
hresholds which further improved with single administration ofhe sEHI TPPU (Fig. 4). Rats fed an omega-6 control diet displayed
odest changes at a low dose of the inhibitor but the DHA dieted rats scored up to 24% higher (insert Fig. 4) at the same dose.hus, with omega-3 dietary supplementation there is a potentialo improve pain relief or potentially dose limit sEHI for long termse in relieving chronic painful conditions.
The sEHI also induce a conditioned place preference (CPP) iniabetic mice [108]. The CPP assay uses conditioning with drugaired environmental cues to assess a preference for context associ-ted pain relief. The CPP assay is believed to assess the non-evokedonic pain typical of chronic pain conditions [109,110]. sEHI induce
robust CPP in neuropathic but not naïve mice which is expectedecause inhibitors do not typically show activity in the absencef a painful state. The CPP assay also demonstrates the sEHI doot have rewarding side effects common of addictive analgesicsespite their efficacy against pain. These results argue that inan sEHI could be powerful and non-addictive pain therapeu-
ics.Inhibition of sEH is also effective against equine laminitis (Fig. 5),
chronic pain condition that often leads to euthanasia of afflictedorses [111]. The result in a clinical patient demonstrates the activ-
ty of the sEHI in a complex setting and above the standard ofare treatment. Importantly, the efficacy of sEH inhibition extendseyond rodents to multiple species including canine and felineork in preparation for publication. Table 2 summarizes the major
utcomes of the nociceptive experiments using the sEHI to elevateultiple classes of EpFA outlined in these sections.
.4. Other biologies of sEHI
The sEH enzyme is present in multiple tissues and cell typesncluding neurons. In the mouse brain sEH immunoreactivity haseen found localized to neurons in the cerebral cortex [112],rigeminal and sphenopalatine ganglia [98]. sEH, which modu-ates EpFAs, also has a role in axonal growth in neurons. Inhibitionf sEH increases axonal outgrowth alone or in combination withETs [113]. This includes the use of multiple structurally unrelatednhibitors which repeat the results observed with direct appli-ation of EETs. The ability to reproduce the effects of the EETsupports targeting the sEH to induce the effects of the EpFAs.dditionally, the increased axonal outgrowth ranges from DRG toiverse neuronal subtypes including cortical neurons. InhibitingEH also effectively delays the onset and reduces the lethality ofABA antagonist induced seizures in mice [100]. The sEHI medi-ted delay in seizure response suggests the brain penetration ofEHI barring blood flow changes. These effects are partially blockedy the steroid synthesis inhibitor finasteride and are enhancedy the neurosteroid allopregnanolone which supports earlier evi-ence that the action of EpFAs may be related to neurosteroid
enesis. Thus, inhibition of sEH is a novel strategy to increase lev-ls of both omega-6 and omega-3 derived EpFAs to mediate botheripheral and CNS mediated analgesia and address additional CNSathologies.was both standing well (Post 9 Days of Treatment) and able to walk around the stall.Treatment of equine laminitis has continued with success and no observed adversereactions to date.
5. Translational pathways
The ultimate purpose of this basic research in nociception andlipid mediators of this biology is to find ways to improve human andanimal health. There is now a substantial amount of evidence thatlong chain fatty acids and their metabolites are potent mediators ofanalgesia. The path forward to translate this evidence into improv-ing human health is determining the best strategy of intervention.Several strategies have been outlined here. Dietary supplemen-tation of omega-3 fatty acids is a safe alternative to improvingnociceptive outcomes. However, there are issues with patient com-pliance in taking daily capsules as well as an aversion to the odorof the typically fish sourced material and gastrointestinal effectsin some patients. Currently there is also very little standardiza-tion of nutritional supplements of omega-3 fatty acids and storageand handling are very important to the stability of the product.Another strategy is the dosing of the EpFAs themselves as they havebeen shown to be active metabolites mediating analgesia. Whilethe administration of EpFAs elicits rapid antihyperalgesia, their usemay be limited to clinical settings. EpFAs are rapidly hydrolyzed atthe pH of stomach acid but they can be administered readily bysimple enteric coating. They would likely be metabolized whilecrossing the intestinal epithelium by the sEH so EpFAs are less
suited to enteral administration and would most often need to beinjected. They are also natural products which makes proprietarycoverage difficult. Synthetic mimics of EpFAs may provide an alter-native solution to the lability of EpFAs, however, their improved
10 K. Wagner et al. / Prostaglandins & other Lipid Mediators 113–115 (2014) 2–12
Table 2Major outcomes of sEHI administration in experimental models.
Pain model Reference sEHI route Species Major outcomes
Inflammatory Inceoglu et al.[87]
Dermal Rat Analgesic against thermal withdrawal LPS model
Inceoglu et al.[36]
s.c.1
i.t.2Rat Analgesic against thermal withdrawal LPS model1, mechanical
allodynia assay CAR model2, and mechanical allodynia diabeticneuropathy model1
Hwang et al.[106]
s.c. Rat Analgesic against mechanical allodynia LPS model
Inceoglu et al.[101]
s.c. Rat Analgesic against PGE2 induced pain
Neuropathy Inceoglu et al.[107]
s.c. Rat Analgesic against mechanical allodynia diabetic neuropathymodel
Wagner et al.[105]
s.c. Rat Analgesic in mechanical allodynia LPS model and diabeticneuropathy model
Wagner et al.[108]
s.c. Mouse Analgesic in mechanical allodynia and conditioned placepreference diabetic neuropathy model
Guedes et al.[111]
i.v. Horse Analgesic in clinical chronic laminitis
Fig. 4 DHA diet + sEHI s.c. Rat Increased analgesia against mechanical allodynia diabeticneuropathy model compared to control supplemented rats
se a sure a su
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.c., subcutaneous; i.v., intravenous; PGE2, prostaglandin E2.1 Corresponds to the LPS model noted to the right in the “Major outcomes” wher2 Corresponds to the CAR model noted to the right in the “Major outcomes” whe
tability and resistance to metabolic degradation may in fact limitheir safety for use in vivo. Future investigations will determinehether these novel molecules have potential for use in animals oran. The strategy that perhaps holds the most promise at least in
he short term is inhibition of the soluble epoxide hydrolase whichlevates the endogenous EpFAs. The EpFAs are normally turnedver very rapidly by sEH which is the major path of their degra-ation. Small molecule inhibitors of sEH elicit potent analgesia at
ow doses in both acute and chronic painful conditions. They haveemonstrated antihyperalgesic activity in experimental species asutlined above, they have favorable pharmacokinetics in dogs [114]nd non-human primates [115] and they have successfully treatedlinical cases of equine laminitis [111]. The efficacy of sEH inhi-ition in these species is attractive given expansion of the animalealth market from industrial production to high value companionnimals and the greater attention paid to chronic painful conditionsuch as osteoarthritis in dogs and cats. Given the toxicity associatedith cyclooxygenase inhibitors in companion animals, the use of
EHI alone or in combination with NSAIDs and coxibs is attractive.oreover, the success of sEHI in multiple species suggests they may
ave success in humans as well. There is one additional strategyemonstrated here (Fig. 4) which is to combine omega-3 supple-entation with administration of sEHI. The omega-3 derived EDPs
re the most potent of the EpFAs and they are also the best sub-trates for sEH [32]. Therefore combining dietary supplementationith sEH inhibition is a logical approach to eliciting the greatest
enefit while limiting the risk of pharmaceutical intervention inreating chronic pain.
. Conclusion
Early research regarding the antinociceptive effects of EpFAsocused on the EETs as the major bioactive metabolites stabi-ized by sEH inhibition. It is now becoming apparent that themega-3 epoxy fatty acid metabolites and specifically EDPs aremportant mediators of this activity. The ability to supplement dieto affect nociception is a promising and safe alternative to currentharmaceutical therapeutics. Moreover, the ability to improve on
ietary supplementation with inhibitors of sEH is a new strategy tontervene in painful conditions with demonstrated effects in bothcute and chronic pain settings. In the future dietary supplemen-ation and sEHI or their combination may be novel therapeutics
perscript appears.perscript appears.
for multiple CNS disorders including pain, and possibly seizures,depression and schizophrenia in man.
Funding
This work was supported by the National Institute of Envi-ronmental Health Sciences (NIEHS) Grant R01 ES002710, NIEHSSuperfund Research Program P42 ES004699, National Instituteof Arthritis and Musculoskeletal and Skin Diseases (NIAMS)R21AR062866, National Institute of Neurological Disorders andStroke (NINDS) U54 NS079202-01 and Grants NIEHS T32ES007059and NIH 5T32DC008072-05 (to K.W.). The content is solely theresponsibility of the authors and does not necessarily represent theofficial views of the National Institutes of Health. B.D.H. is a Georgeand Judy Marcus Senior Fellow of the American Asthma Foundation.
Conflict of interest
The University of California holds patents on the sEH inhibitorsused in this study as well as their use to treat inflammation, inflam-matory pain, and neuropathic pain. B.D. Hammock and B. Inceogluare co-founders of Eicosis L.L.C., a startup company advancing sEHinhibitors into the clinic. K. Wagner and S. Vito have no conflicts todeclare.
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